School Of Basic And Applied Sciences
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Item Structural, kinetic and thermodynamic characterizations of SDS-induced molten globule state of a highly negatively charged cytochrome c(Oxford University Press, 2019) Jain, R; Sharma, D; Kumar, Rakesh; Kumar, RajeshThis study presents the structural, kinetic and thermodynamic characterizations of previously unknown submicellar concentrations of SDS-induced molten globule (MGSDS) state of a highly negatively charged basedenatured ferricytochrome c (U B -state) at pH ∼12.8 (±0.2). The far-UV CD, near-UV CD, ANS-fluorescence data of UB-state in the presence of different concentrations of SDS indicate that the submicellar concentrations of SDS (≤0.4mM) transform the UBstate to MG SDS -state. The MG SDS -state has nativelike α-helical secondary structure but lacks tertiary structure. The free energy change (ΔG° D) for U B → MG SDS transition determined by far-UV CD (∼2.7 kcal mol -1 ) is slightly higher than those determined by fluorescence (∼2.0 kcal mol -1 ) at 25°C. At very low SDS and NaCl concentrations, the MG SDS -state undergoes cold denaturation. As SDS concentration is increased, the thermal denaturation temperature increases and the cold denaturation temperature decrease. Kinetic experiments involving the measurement of the CO-association rate to the base-denatured ferrocytochrome c at pH ≈12.8 (±0.2), 25°C indicate that the submicellar concentrations of SDS restrict the internal dynamics of base-denatured protein. © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.Item Factors defining the effects of macromolecular crowding on dynamics and thermodynamic stability of heme proteins in-vitro,(Elsevier, 2018) Kumar, Rajesh; Sharma, Deepak; Kumar, Vinay; Kumar, RajeshThe role of crowding agents on structure and activities of heme proteins has been established. Analysis of kinetic and thermodynamic parameters measured for CO-dissociation reaction of natively-folded carbonmonoxycytochrome c (NCO) and carbonmonoxymyoglobin (MbCO) at different [GdnHCl] or [Urea] in the presence of crowding agents (dextran 40, dextran 70 and ficoll 70) demonstrate that (i) at low denaturant concentrations, crowder presence enhances the denaturant-mediated restricted dynamics of NCO and MbCO, and (ii) at higher denaturant concentrations, large scale unfolding-fluctuations dominate the dynamics and inclusion of crowder counteracts the structural-fluctuations causing the unfolding of proteins. Thermodynamic analysis of thermal and urea-unfolding curves of cytochrome c (Cyt c) and myoglobin (Mb) measured at different [GdnHCl] in presence of crowding agents reveals that crowder presence counterbalances and strengthens the destabilizing action of GdnHCl on stability of Cyt c and Mb, respectively. This study further demonstrates that the size, shape and concentration of crowding agent modulate the effect of crowder on denaturant-mediated dynamics and thermodynamic stability of heme proteins.Item Macromolecular crowding-induced molten globule states of the alkali pH-denatured proteins(Elsevier B.V., 2018) Kumar, Rajesh; Kumar, Rajesh; Sharma, Deepak; Garg, Mansi; Kumar, Vinay; Agarwal, Mukesh ChandStructural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pH 12.9 (±0.1) to molten globule (MG) states (C B -states). These results further revealed that the C B -states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pH 12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing ?-loop that control CO association to alkaline Ferrocyt c. - 2018 Elsevier B.V.Item Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c(Academic Press Inc., 2016) Kumar, RajeshThis paper analyzes the effect of pH on thermodynamic stability, low-frequency local motions and microsecond folding kinetics of carbonmonoxycytochrome c (Cyt-CO) all across the alkaline pH-unfolding transition of protein. Thermodynamic analysis of urea-induced unfolding transitions of Cyt-CO measured between pH 6 and pH 11.9 reveals that Cyt-CO is maximally stable at pH?9.5. Dilution of unfolded Cyt-CO into refolding medium forms a native-like compact state (NCO-state), where Fe2+?CO interaction persists. Kinetic and thermodynamic parameters measured for slow thermally-driven CO dissociation (NCO?N+CO) and association (N+CO?NCO) reactions between pH 6.5 and pH 13 reveal that the thermal-motions of M80-containing ?-loop are decreased in subdenaturing limit of alkaline pH. Laser photolysis of Fe2+-CO bond in NCO-state triggers the microsecond folding (NCO?N). The microsecond kinetics measured all across the alkaline pH-unfolding transition of Cyt-CO produce rate rollover in the refolding limb of chevron plot, which suggests a glass transition of NCO en route to N. Between pH 7 and pH 11.9, the natural logarithm of the microsecond folding rate varies by?<?1.5 units while the natural logarithm of apparent equilibrium constant varies by 11.8 units. This finding indicates that the pH-dependent ionic-interactions greatly affect the global stability of protein but have very small effect on folding kinetics. ? 2016 Elsevier Inc.Item Role of Macromolecular Crowding on Stability and Iron Release Kinetics of Serum Transferrin(American Chemical Society, 2017) Kumar, Sandeep; Sharma, Deepak; Kumar, RajeshThe macromolecular crowding influences the structural stability and functional properties of transferrin (Tf). The equilibrium as well as kinetic studies of Tf at different concentrations of crowding agents (dextran 40, dextran 70, and ficoll 70) and at a fixed concentration of dextran 40 under different concentrations of NaCl at pH 7.4 and 5.6 (?1) revealed that (i) the crowder environment increases the diferric-Tf (Fe2Tf) stability against iron loss and overall denaturation of the protein, (ii) both in the absence and presence of crowder, the presence of salt promotes the loss of iron and overall denaturation of Fe2Tf which is due to ionic screening of electrostatic interactions, (iii) the crowder environment retards iron release from monoferric N-lobe of Tf (FeNTf) by increasing enthalpic barrier, (iv) the retardation of iron release by crowding is enthalpically dominated than the entropic one, (v) both in the absence and presence of crowder, the presence of salt accelerates the iron release from FeNTf due to ionic screening of electrostatic interactions and anion binding to KISAB sites, and (vi) the crowders environment is unable to diminish (a) the salt-induced destabilization of Fe2Tf against the loss of iron and overall denaturation and (b) the anion effect and ionic screening of diffusive counterions responsible to promote iron release from FeNTf. ? 2017 American Chemical Society.